Studies to find new-unknown blood borne viruses (BBV) generally focus on individual blood samples of donors or recipients with clinical symptoms (fever, hepatitis, etc.). Patients with hemophilia, sickle cell disease, thalassemia, agammaglobulinemia, transplant recipients and others, require chronic transfusion of blood or blood derived products, often made by pooling samples of 20-60,000 blood donors. Given this fact, we hypothesize that an unbiased high- throughput sequencing (UHTS) based study of a few hundred multiple transfusion recipients (MTR) will enable identification of several new BBV that otherwise can only be found by screening several thousands of individual blood samples. Multicenter Hemophilia Cohort Study (MHCS) subjects received multiple transfusions of coagulation factors made by pooling plasma of thousands of individual donors. Moreover, a majority of MHCS subjects are co-infected with HIV, making them highly susceptible to secondary virus infections; therefore we anticipate that the BBVs that are very rare infections in general population will be common infections in MHCS subjects. To test these hypothesis, we used UHTS (preliminary results) to analyze two samples each of 16 MHCS subjects separated by >4000 days (>10 yrs.). We detected an average of 3.4 and 10.4 different viruses in first and last time point samples, respectively. Evolutionary analyses done using the species specific PCR and clonal sequencing indicated distinct genetic diversity, composition and co-variance of different BBV species over time. Noticeably, in addition to finding high prevalence of HIV, HCV, HBV, GBVc, TTV and Parvovirus B19, we found several recently identified viruses never before reported in human blood samples and several new viruses. Our goal is genetic characterization of these new BBV found in human serum samples, confirm their authentic human infection using serology and determine their infection prevalence in different MTR cohorts, Transfusion Transmitted Virus Study subjects, healthy blood donors and other disease cohorts. Approximately 5 million individuals require transfusion of human blood derived product every year in the United States alone. The proposed identification of new viruses found in human blood, their genetic characterization and prevalence in different population will help in conducting subsequent studies to determine their risk of transfusion transmission and in developing strategies to prevent new infections/diseases.